Compositions and methods for achieving sustained therapeutic drug concentrations in a subject

ABSTRACT

Provided herein are compounds and methods for achieving a sustained therapeutic effect of small molecule anti-cancer agents when administered in vivo.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 application of InternationalApplication No. PCT/US2009/005284, filed Sep. 23, 2009, designating theUnited States, which claims the benefit of priority under 35 U.S.C.§119(e) to the following U.S. Provisional Patent Applications: U.S.Provisional Patent Application No. 61/099,516 filed 23 Sep. 2008; U.S.Provisional Patent Application No. 61/106,931 filed 20 Oct. 2008; andU.S. Provisional Patent Application No. 61/173,433 filed 28 Apr. 2009,all of which are incorporated herein by reference in their entireties.

FIELD

This invention relates to compounds and methods for achieving asustained therapeutic effect of small molecule anti-cancer agents whenadministered in vivo.

BACKGROUND

Over the years, numerous methods have been proposed for improving thedelivery of biologically active agents, and in particular, smallmolecule anticancer compounds. Challenges associated with theformulation and delivery of cancer chemotherapeutics can include pooraqueous solubility, toxicity, low bioavailability, instability, andrapid in-vivo degradation, to name just a few. Although many approacheshave been devised for improving the delivery of anticancer compounds, nosingle approach is without its significant drawbacks. For instance,commonly employed drug delivery approaches aimed at solving or at leastameliorating one or more of these problems include drug encapsulation,such as in a liposome, polymer matrix, or unimolecular micelle, covalentattachment to a water-soluble polymer such as polyethylene glycol, useof gene targeting agents, nanoparticles, and the like.

The clinical effectiveness of many small molecule therapeutics, andoncolytics in particular, is limited by several factors. For instance,irinotecan and other camptothecin derivatives undergo an undesirablehydrolysis of the E-ring lactone under alkaline conditions.Additionally, administration of irinotecan causes a number of troublingside effects, including leucopenia, neutropenia, and diarrhea. Due toits severe diarrheal side-effect, the dose of irinotecan that can beadministered in its conventional, unmodified form is extremely limited,thus hampering the efficacy of this drug and others of this type. Suchharsh side effects, when severe, can be sufficient to arrest furtherdevelopment of such drugs as promising therapeutics. Additionalchallenges facing small molecule oncolytics include high clearance ratesand minimal tumor permeation and residence time. Indeed, manychemotherapies are often accompanied by ultimate failure. Thus, thedesign and development of biocompatible delivery systems for anticancercompounds, as well as related therapeutic methods continues to present asignificant challenge. Such challenge is met by the compounds andmethods provided herein.

SUMMARY

In one aspect, the present disclosure is based at least in part upon theApplicant's surprising discovery of small molecule anticancer prodrugsthat are effective to release an active moiety in vivo, wherein theeffective half life in humans of the active moiety is greater than 50hours.

In one embodiment of the foregoing, the prodrug effectively achievesmetronomic dosing in vivo upon a single administration. Illustrativeexamples of a single administration include parenteral administration(including intraperitoneal, intravenous, subcutaneous, or intramuscularinjection), oral, rectal, topical, nasal, and ophthalmic administrationas well as an infusion with a pump over, for example, a period of 30 to120 minutes.

Thus, provided herein is a method of achieving a metronomic dosingprofile in vivo upon a single administration of a topoisomeraseinhibitor prodrug. The method comprises administering a prodrug of atopoisomerase inhibitor to a mammalian subject (such as a human),wherein the topoisomerase inhibitor is releasably attached to apolyethylene glycol moiety, to thereby achieve in a single dose of theprodrug, plasma levels of the topoisomerase inhibitor or a metabolitethereof that remain above the level of detection for at least seven dayspost administration.

In one embodiment, plasma levels of the topoisomerase inhibitor or ametabolite thereof that remain above a level of detection that is atabout 0.5 ng/mL.

In yet another embodiment, plasma levels of the topoisomerase inhibitoror a metabolite thereof that remain above a level of detection that isat about 1.0 ng/mL.

In yet another embodiment, plasma levels of the topoisomerase inhibitoror a metabolite thereof that remain above a level of detection that isat about 2.0 ng/mL.

In a related embodiment, the method or use of the prodrug is effectiveto achieve in a single dose of the prodrug, plasma levels of thetopoisomerase inhibitor or a metabolite thereof that remain at leastabout two times above the level of detection for at least seven dayspost administration, or even remain at least about three times above thelevel of detection for at least seven days post administration.

In certain embodiments, the method or use of the prodrug is effective toachieve in a single dose of the prodrug, plasma levels of thetopoisomerase inhibitor or a metabolite thereof that remain above thelevel of detection for at least 21 days post administration.

In yet one or more further embodiments, the method is effective othereby achieve in a single dose of the prodrug, plasma levels of thetopoisomerase inhibitor or a metabolite thereof that remain above thelevel of detection for at least seven days post administration, such asachieved by daily metronomic dosing of the topoisomerase inhibitor at adaily dosage amount that is at least two times lower than the dosageamount of topoisomerase inhibitor administered in prodrug form in theadministering step.

In one or more embodiments related to the foregoing, the mode ofadministering is selected from intraperitoneal, intravenous,subcutaneous, and intramuscular injection.

In one exemplary embodiment, the prodrug is of a topoisomerase inhibitorsuch as but not limited to irinotecan, topotecan, camptothecin, orlamellarin D.

In yet another exemplary embodiment, the prodrug is of a microtubuleinhibitor such as but not limited to vincristine, vinblastine,vinflunine, and docetaxel.

In yet a further embodiment, the prodrug is an irinotecan prodrug.

In yet another embodiment, the prodrug is multi-armed.

In a more specific embodiment, the multi-armed prodrug possesses from3-10 polyethylene glycol arms each having a topoisomerase inhibitormolecule releasably attached thereto.

In an even more particular embodiment of any one or more of theforegoing, the prodrug has the structure:

In yet an additional embodiment directed to administering a prodrug ofirinotecan as described herein, the method is effective to achieve viathe administering step, plasma levels of irinotecan or a metabolitethereof that remain above about 0.2 ng/mL for at least seven days postadministration

In a further embodiment, the prodrug, when administered in vivo,provides a desired concentration-time profile to achieve a desiredpharmacodynamic effect, wherein the desired pharmacodynamic effect isdifferent from the known (or understood or accepted) pharmacodynamiceffect of the active moiety.

In a related embodiment of the foregoing method, the prodrug is ofirinotecan, where the desired pharmacodynamic effect isantiangiogenesis, and the known pharmacodynamic effect is topoisomeraseinhibition. In yet another related embodiment, the prodrug is ofdocetaxel, where the desired pharmacodynamic effect is antiangiogenesis,and the known pharmacodynamic effect is microtubule inhibition.

In yet another embodiment, the effective half life in humans of theactive moiety is selected from the group consisting of half livesgreater than 7 days, 10 days, 14 days, 20 days, 21 days, 25 days, 28days, 30 days, 35 days, 40 days, 45 days, 49 days, 50 days, 60 days, 70days, 80 days, 90 days, and 100 days.

In yet another particular embodiment, the prodrug is of a small moleculeselected from the group consisting of platins, oxymorphone analogues,steroids, quinolones, and nucleosides.

In yet a further and more particular embodiment, the prodrug is of thesmall molecule, irinotecan,

In a second aspect, provided herein is a method of achieving sustainedexposure to SN-38 via administration of irinotecan to a mammaliansubject. The method comprises administering via a non-continuous dosingregimen to a mammalian subject having one or more cancerous solidtumors, a therapeutically effective amount of a pharmaceuticalcomposition comprising a prodrug corresponding to structure (I):

wherein the non-continuous dosing regimen comprises administering thepharmaceutical composition no more frequently than once every sevendays, to thereby maintain sustained therapeutic levels of SN-38 inplasma between dosings.

In one embodiment, the therapeutic levels of SN-38 remain at or above aplasma concentration of about 0.2 ng/mL.

In one or more additional embodiments, the therapeutic levels of SN-38remain at or above a plasma concentration selected from the groupconsisting of about 0.3 ng/mL, about 0.4 ng/mL, and about 0.5 ng/mL.

In yet a further embodiment, the dosing regimen comprises administeringthe pharmaceutical composition once every 21 days and the therapeuticlevels of SN-38 remain at or above a plasma concentration of about 0.4ng/mL between dosings.

In a third aspect, provided herein is a method for achieving extendedtherapeutic efficacy of irinotecan upon administration. The methodcomprises administering to a mammalian subject having one or morecancerous solid tumors, a therapeutically effective amount of apharmaceutical composition comprising a prodrug corresponding tostructure (I):

wherein the administering comprises administering the composition at afrequency of between once every 7 days to once every 30 days, to therebyachieve an elimination half-life in plasma of SN-38 that exceeds 750hours.

In one embodiment directed at least to the third aspect, the eliminationhalf-life in plasma of SN-38 exceeds 900 hours.

In yet a further embodiment, the elimination half-life in plasma ofSN-38 exceeds 1000 hours.

In yet another embodiment, the elimination half-life in plasma of SN-38exceeds 1100 hours.

The following embodiments are directed to the foregoing second and thirdaspects as set forth above.

In one embodiment, the overall nominal average molecular weight of theprodrug ranges from about 10,000 to about 60,000 daltons. For example,the overall nominal average molecular weight of the prodrug is selectedfrom about 10,000 daltons, 20,000 daltons, 30,000 daltons, 40,000daltons, 50,000 daltons and 60,000 daltons.

In yet another embodiment, dosing occurs at a frequency selected fromonce every 7 days, once every 14 days, once every 21 days and once every28 days.

In additional embodiments, the solid tumor type is selected fromovarian, breast, cervical, maxillary sinus, bladder, colorectal, smallcell lung, and non-small cell lung.

In yet an additional embodiment, the administering comprisesadministering to the subject a dosage amount of irinotecan ranging fromabout 70 mg/m² to about 300 mg/m².

In yet another embodiment, the administering is intravenous.

In yet a further embodiment, the administering is effective to preventtumor growth as measured from the start of treatment.

In yet an additional embodiment, the administering is effective toresult in tumor size regression.

In a related embodiment, the administering is effective to result intumor size regression of at least 20%.

In yet another aspect, provided herein is a method for treating a humansubject having a cancer that was refractory to treatment with one ormore anticancer agents, by administering a therapeutically effectiveamount of a topoisomerase inhibitor prodrug as described herein.

In a related embodiment, the human subject is one whose cancer waspreviously resistant to treatment with 1, or 2, or 3, or 4, or 5 or moreanticancer agents. That is to say, in prior treatments, progression ofthe cancer was observed, while upon administration of a therapeuticallyeffective amount of an exemplary prodrug, e.g., in one embodiment,compound I, anti-tumor activity was observed, either by virtue ofpartial tumor regression, arrestment of tumor growth, or by evidenceprovided by one or more biomarkers.

Additional methods include treatment of (i) metastatic breast cancerthat is resistant to anthracycline and/or taxane based therapies, (ii)platinum-resistant ovarian cancer, (iii) metastatic cervical cancer, and(iv) colorectal cancer in patients with K-Ras mutated gene status byadministering a therapeutically effective amount of a prodrug of atopoisomerase inhibitor molecule as described herein.

In one embodiment, the prodrug is one in which a topoisomerase inhibitoris releasably attached to a polyethylene glycol moiety.

In yet another aspect, provided is a method for treating metastaticbreast cancer in which a topoisomerase inhibitor prodrug as describedherein is administered to a patient with locally advanced metastaticbreast cancer at a therapeutically effective amount, where the patienthas had no more than two prior (unsuccessful) treatments withanthracycline and/or taxane based chemotherapeutics.

In yet a another aspect, provided is a method for treating platinumresistant ovarian cancer. In the method, a topoisomerase inhibitorprodrug as described herein is administered to a patient with locallyadvanced or metastatic ovarian cancer at a therapeutically effectiveamount, where the patient has shown tumor progression duringplatinum-based therapy. In one particular embodiment, the patient hashad a progression-free interval of less than six months.

An exemplary compound for use in any one or more of the methodsdescribed herein corresponds to structure I.

In yet another approach, a topoisomerase inhibitor prodrug as providedherein (e.g., such as that in Example 1) is administered to a subjectwith locally advanced colorectal cancer, where the colorectal tumor(s)has a K-Ras oncogene mutation (K-Ras mutant types) such that the tumordoes not respond to EGFR-inhibitors, such as cetuximab. Subjects arethose having failed one prior 5-FU containing therapy, and are alsoirinotecan naïve.

Also provided are uses of the subject prodrugs in any one or more of theabove described methods.

Additional embodiments of the present prodrugs, related compositions andmethods will be apparent from the following description, drawings andexamples. As can be appreciated from the foregoing and followingdescription, each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present disclosure provided that the features included insuch a combination are not mutually inconsistent. In addition, anyfeature or combination of features may be specifically excluded from anyembodiment of the present disclosure.

These and other objects and features will become more fully apparentwhen read in conjunction with the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating sustained exposure to the activemetabolite of irinotecan, SN-38, in human subjects administered aprodrug of irinotecan (“Compound I”) on a 21 day dosing schedule (solidlines) in comparison to irinotecan (Camptosar®) as described in detailin Example 2. Specifically, the plot illustrates simulated SN-38concentrations (ng/mL) over time (weeks) based upon the pharmacokineticstudy described in Example 2.

FIG. 2 is a graph illustrating median tumor volume (mm³) versus dayspost initial treatment in a platinum resistant 2780 ovarian cancer modelas described in detail in Example 3. Figure Legend: Closed circles,black (●): no treatment (top data plot)—endpoint reached prior to 20days post-treatment; open circles (∘): treatment with cisplatin, topplot next to the “no treatment” data, endpoint reached prior to 20 dayspost treatment; open triangles (Δ): treatment with carboplatin, plotnearly identical to cisplatin data, endpoint reached prior to 20 dayspost treatment; next three plots in center of graph—irinotecan data atcenter portion of graph, administered at varying doses: (closedtriangles, grey (▾), 50 mg/kg irinotecan; closed circles, grey (●), 100mg/kg irinotecan; and (closed upwards triangles, grey ▴), 150 mg/kgirinotecan—all reaching endpoint at approximately 30 days post initialtreatment; final three plots of Compound I administered at varyingdoses, closed triangles, grey (▾), 50 mg/kg Compound I; closed circles,grey (●), 100 mg/kg Compound I; and closed upwards triangles, grey (▴),150 mg/kg Compound I, all extending to greatest number of days to reachendpoint, e.g., approximately 50 days.

FIG. 3 is a table summarizing the agent administered, treatment regimen,route of administration, dosing schedule, tumor growth delay response,and other data related to the platinum resistant ovarian 2780 ovariancancer model described above and in Example 3.

DETAILED DESCRIPTION

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

DEFINITIONS

It must be noted that, as used in this specification, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to a “polymer” includesa single polymer as well as two or more of the same or differentpolymers, reference to a “conjugate” refers to a single conjugate aswell as two or more of the same or different conjugates, reference to an“excipient” includes a single excipient as well as two or more of thesame or different excipients, and the like.

The following terminology will be used in accordance with thedefinitions described below.

“PEG” or “poly(ethylene glycol)” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Typically, PEGs for use in thepresent invention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. The variable (n) is 3 to 3000, and theterminal groups and architecture of the overall PEG may vary. When PEGor a conjugate comprising a PEG segment further comprises a spacer or alinker as in structure I above (to be described in greater detailbelow), the atoms comprising the spacer (X) or linker (Q), whencovalently attached to a PEG segment, do not result in formation of (i)an oxygen-oxygen bond (—O—O—, a peroxide linkage), or (ii) anitrogen-oxygen bond (N—O, O—N). PEGs for use in the invention includePEGs having a variety of molecular weights, structures or geometries tobe described in greater detail below.

“Water-soluble”, in the context of a polymer of the invention or a“water-soluble polymer segment” is any segment or polymer that issoluble in water at room temperature. Typically, a water-soluble polymeror segment will transmit at least about 75%, more preferably at leastabout 95% of light, transmitted by the same solution after filtering. Ona weight basis, a water-soluble polymer or segment thereof willpreferably be at least about 35% (by weight) soluble in water, morepreferably at least about 50% (by weight) soluble in water, still morepreferably about 70% (by weight) soluble in water, and still morepreferably about 85% (by weight) soluble in water. It is most preferred,however, that the water-soluble polymer or segment is about 95% (byweight) soluble in water or completely soluble in water.

“Molecular mass” in the context of a water-soluble polymer of theinvention such as PEG, refers to the nominal average molecular mass of apolymer, typically determined by size exclusion chromatography, lightscattering techniques, or intrinsic viscosity determination in water ororganic solvents. Molecular weight in the context of a water-solublepolymer, such as PEG, can be expressed as either a number-averagemolecular weight or a weight-average molecular weight. Unless otherwiseindicated, all references to molecular weight herein refer to thenumber-average molecular weight. Both molecular weight determinations,number-average and weight-average, can be measured using gel permeationchromatographic techniques. Other methods for measuring molecular weightvalues can also be used, such as the use of end-group analysis or themeasurement of colligative properties (e.g., freezing-point depression,boiling-point elevation, or osmotic pressure) to determinenumber-average molecular weight or the use of light scatteringtechniques, ultracentrifugation or viscometry to determineweight-average molecular weight. The polymers of the invention aretypically polydisperse (i.e., number-average molecular weight andweight-average molecular weight of the polymers are not equal),possessing low polydispersity values such as less than about 1.2, lessthan about 1.15, less than about 1.10, less than about 1.05, and lessthan about 1.03. As used herein, references will at times be made to asingle water-soluble polymer having either a weight-average molecularweight or number-average molecular weight; such references will beunderstood to mean that the single-water soluble polymer was obtainedfrom a composition of water-soluble polymers having the stated molecularweight.

“Multi-armed” in reference to the geometry or overall structure of apolymer refers to polymer having 3 or more polymer-containing “arms”connected to a “core” molecule or structure. Thus, a multi-armed polymermay possess 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymerarms, 7 polymer arms, 8 polymer arms or more, depending upon itsconfiguration and core structure. One particular type of highly branchedpolymer is a dendritic polymer or dendrimer, that, for the purposes ofthe invention, is considered to possess a structure distinct from thatof a multi-armed polymer. That is to say, a multi-armed polymer asreferred to herein explicitly excludes dendrimers.

A “dendrimer” is a globular, size monodisperse polymer in which allbonds emerge radially from a central focal point or core with a regularbranching pattern and with repeat units that each contribute a branchpoint. Dendrimers are typically formed using a nano-scale, multistepfabrication process. Each step results in a new “generation” that hastwo or more times the complexity of the previous generation. Dendrimersexhibit certain dendritic state properties such as core encapsulation,making them unique from other types of polymers.

“Branch point” refers to a bifurcation point comprising one or moreatoms at which a polymer splits or branches from a linear structure intoone or more additional polymer arms. A multi-arm polymer may have onebranch point or multiple branch points, so long as the branches are notregular repeats resulting in a dendrimer.

“Active agent” as used herein includes any agent, drug, compound, andthe like which provides some pharmacologic, often beneficial, effectthat can be demonstrated in-vivo or in vitro. As used herein, theseterms further include any physiologically or pharmacologically activesubstance that produces a localized or systemic effect in a patient.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of an active agent present in a pharmaceuticalpreparation that is needed to provide a desired level of active agentand/or conjugate in the bloodstream or in a target tissue or site in thebody. The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofpharmaceutical preparation, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

The terms “subject”, “individual” or “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, murines, rodents, simians, humans, farm animals,sport animals and pets. Such subjects are typically suffering from orprone to a condition that can be prevented or treated by administrationof a polymer of the invention, typically but not necessarily in the formof a polymer-active agent conjugate as described herein.

“Metronomic” dosing refers to administration of comparatively low losesof drug, typically a chemotherapeutic, on a frequent or continuousschedule, with no extended interruptions. Generally, a metronomic doseis a lower dose than that administered using conventional therapy, e.g.,a dose that is about 10% to about 75% of the conventional dose, and ismore typically around 10% to about 50% of a recommended doseadministered using conventional therapy, when calculated to a dailydosage amount. Metronomic dosing typically involves daily oral andcontinuous infusion schedules.

The term “about”, particularly in reference to a given quantity, ismeant to encompass deviations of plus or minus five percent.

“Treatment” or “treating” of a particular condition includes: (1)preventing such a condition, i.e. causing the condition not to develop,or to occur with less intensity or to a lesser degree in a subject thatmay be exposed to or predisposed to the condition but does not yetexperience or display the condition, (2) inhibiting the condition, i.e.,arresting the development or reversing the condition.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

A “small molecule” may be defined broadly as an organic, inorganic, ororganometallic compound typically having a molecular weight of less thanabout 1000. Small molecules of the invention encompass oligopeptides andother biomolecules having a molecular weight of less than about 1000.

Small Molecule Anti-Cancer Prodrugs

As described generally above, the instant disclosure relates to smallmolecule anticancer prodrugs. A prodrug refers to a compound that isgenerally (but not necessarily) inactive and in a form altered from theactive parent drug, where upon administration, the prodrug ismetabolized in vivo into an active metabolite. Preferably, the prodrugsprovided herein are water-soluble polymer conjugates of small moleculeanticancer agents.

In one particular embodiment, the prodrug is a multi-armed polymerprodrug of a small molecule anticancer agent.

Typically, the total number average molecular weight of the overallmulti-arm polymer portion of a polymer conjugate is about 1,000 daltons(Da) to about 100,000 Da, more preferably about 10,000 Da to about60,000 Da, most preferably about 15,000 to about 60,000 Da. Multi-armedpolymers having a number average molecular weight of about 5,000 Da,about 8,000 Da, about 10,000 Da, about 12,000 Da, about 15,000 Da, about20,000 Da, about 25,000 Da, about 30,000 Da, about 35,000 Da, about40,000 Da, about 45,000 Da, about 50,000 Da, and about 60,000 Da areparticularly preferred. Multi-armed polymers having a molecular weightof 20,000 Da or greater, i.e., of about 20,000 Da, or 25,000 Da, or30,000 Da, or 40,000 Da or 50,000 Da, or 60,000 Da, are particularlypreferred for tumor-targeting applications. The actual molecular weightof the multi-armed polymer will depend, of course, on the number ofpolymer arms and the molecular weight of each polymer arm in the overallmulti-armed polymer.

Since they are prodrugs, in the case of a conjugates such as a watersoluble polymer conjugate, the linkage between the polymer portion andthe small molecule anticancer agent is preferably hydrolyticallydegradable for in vivo release of the parent drug molecule over time.Representative hydrolytically degradable linkages include carboxylateester, carbonate ester, phosphate ester, anhydride, acetal, ketal,acyloxyalkyl ether, imine, orthoester, and oligonucleotides. Esters suchas carboxylate and carbonate esters are particularly preferred linkages.The particular linkage and linkage chemistry employed will depend uponthe particular small molecule anticancer agent, the presence ofadditional functional groups within the active agent, and the like, andcan be readily determined by one skilled in the art based upon theguidance presented herein.

With respect to the prodrugs provided herein, it is not necessary forthe prodrug itself to exhibit biological activity, since the parent drugis released upon hydrolysis. However, in certain embodiments, theprodrug maintains at least a measurable degree of activity. That is tosay, in some instances, a prodrug possesses anywhere from about 1% toabout 100% or more of the specific activity of the unmodified parentcompound. In a preferred embodiment, a prodrug as provided herein willpossess one or more therapeutic advantages over the unmodified parentdrug. That is to say, a prodrug as provided herein will possess fromabout 1% to about 100% bioactivity relative to the unmodified parentanticancer agent, prior to conjugation. Such activity may be determinedusing a suitable in-vivo or in-vitro model, depending upon the knownactivity of the particular parent compound. For anticancer drugs, invivo anticancer activity is typically evaluated by comparison of growthrates of tumor implants in drug treated and control groups of athymicmice using well-established animal models or in human-based trials asdescribed herein in the accompanying examples. For instance, in certainanimal models, anticancer activity is indicated by slower tumor growthrates in the treated group relative to the control group (J. W. Singer,et al., Ann. N.Y. Acad. Sci., 922: 136-150, 2000). As can be seen In theaccompanying examples, any one of a number of indicators may be employedto demonstrate anticancer activity: e.g., positive response to treatmentas indicated by cessation of tumor growth or tumor size regression,particularly in cases where treatment with either well-established orinvestigational anticancer agents has proven unsuccessful (i.e.,refractory cases of cancer), improved plasma half-lives, greatlyextended/sustained exposure profiles, altered plasma profiles, and thelike.

Additionally, the severity of the side effects associated withadministration of the prodrugs of the invention is preferably comparableto, or even more preferably, is less than, the side effects associatedwith administration of the parent compound. In particular, preferredanticancer prodrugs, particularly those of an anticancer agent such asirinotecan, when administered to a patient, result in reducedleucopenia/neuteopenia and diarrhea when compared to the unmodifiedparent drug molecule. The severity of side effects of anticancer agentssuch as camptothecin and camptothecin-like compounds can be readilyassessed (See, for example, Kado, et al., Cancer Chemotherapy andPharmacology, Aug. 6, 2003). Administration of a prodrug as providedherein will typically result not only in improved efficacy, but also inreduced side effects (e.g., toxicity) such as those described above whencompared to the parent drug.

Structural Features of Prodrugs

Preferred prodrugs are those comprising a multi-arm polymer, i.e.,having three or more arms, and having the generalized structure:R(-Q-POLY₁-X-D)_(q)

as described in detail in Applicant's United States Patent ApplicationNo. 20050112088, the contents of which is hereby incorporated herein inits entirety. One particularly preferred conjugate is described inExamples 1 and 2.

In the above structure, R is an organic radical possessing from about 3to about 150 carbon atoms, preferably from about 3 to about 50 carbonatoms, and even more preferably from about 3 to about 10 carbon atoms,optionally containing one or more heteroatoms (e.g., O, S, or N). In oneembodiment, R possesses a number of carbon atoms selected from the groupconsisting of 3, 4, 5, 6, 7, 8, 9, and 10. R may be linear or cyclic,and typically, emanating therefrom are at least 3 independent polymerarms each having at least one active agent moiety covalently attachedthereto. Looking at the above structure, “q” corresponds to the numberof polymer arms emanating from “R”.

Q is a linker, preferably one that is hydrolytically stable. Typically,Q contains at least one heteratom such as O, or S, or NH, where the atomproximal to R in Q, when taken together with R, typically represents aresidue of the core organic radical R. Illustrative examples areprovided below. Generally, Q contains from 1 to about 10 atoms, or from1 to about 5 atoms. More particularly, Q typically contains one of thefollowing number of atoms: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In aparticular embodiment, Q is O, S, or —NH—C(O)—.

POLY₁ represents a water-soluble and non-peptidic polymer as describedin detail below.

X is a spacer that comprises a hydrolyzable linkage, where thehydrolyzable linkage is attached directly to the active agent, D.Typically, at least one atom of the hydrolyzable linkage is contained inthe active agent, D, in its unmodified form, such that upon hydrolysisof the hydrolyzable linkage comprised within X, the active agent, D, isreleased. Generally speaking, the spacer, X, has an atom length of fromabout 4 atoms to about 50 atoms, or more preferably from about 5 atomsto about 25 atoms, or even more preferably from about 5 atoms to about20 atoms. Representative spacers have a length of from about 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 atoms.

In yet another particular embodiment, X possesses the structure: Y-Z,where Y is a spacer fragment covalently attached to Z, a hydrolyticallydegradable linkage. In certain embodiments, Z itself may not constitutea hydrolytically degradable linkage, however, when taken together withY, or at least a portion of Y, forms a linkage that is hydrolyticallydegradable. Preferably, Y comprises(CH₂)_(a)—C(O)NH—(CH₂)_(0,1)—(CH₂CH₂O)₀₋₁₀.

Preferably, R_(x) and R_(y) in each occurrence are independently H orlower alkyl. In one embodiment, R_(x) and R_(y) are in each occurrenceH. In yet another embodiment, a ranges from 0 to 5. In yet anotherembodiment, b ranges from 0 to 5. In yet another embodiment, c rangesfrom 0 to 10. In yet another embodiment, K is —C(O)—NH. Any of theembodiments described herein is meant to apply not only to generalizedstructure I, but also to extend to particular combinations ofembodiments.

In yet another embodiment, R_(x) and R_(y) in each occurrence are H, ais 1, K is —C(O)—NH, and b is 0 or 1.

Representative examples of X include —CH₂—C(O)—NH—CH₂—C(O)O— (here, Ycorresponds to —CH₂—C(O)—NH—CH₂— and Z corresponds to —C(O)—O—), and—CH₂—C(O)—NH—(CH₂CH₂O)₂—C(O)—O— (here, Y corresponds to—CH₂—C(O)—NH—(CH₂CH₂O)₂— and Z corresponds to —C(O)—O—).

Returning now to the above structure, D is an active agent moiety suchas a small molecule, and q (the number of independent polymer arms)ranges from about 3 to about 50. Preferably, q ranges from about 3 toabout 25. More preferably, q is from 3 to about 10, and possesses avalue of 3, 4, 5, 6, 7, 8, 9, or 10.

In accordance with one embodiment of the invention, the conjugatecomprises a polymer having from about 3 to about 25 small moleculescovalently attached thereto. More particularly, the conjugate comprisesa water soluble polymer having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 active agent moleculescovalently attached thereto. In a further embodiment, the conjugate ofthe invention has from about 3 to about 8 active agent moleculescovalently attached to the water-soluble polymer. Typically, althoughnot necessarily, the number of polymer arms will correspond to thenumber of active agents covalently attached to the water solublepolymer.

In instances in which the prodrug comprises a polymer conjugate, thepolymer portion is preferably a water-soluble and non-peptidic polymer.Any of a variety of polymers that are non-peptidic and water-soluble canbe used to form a conjugate in accordance with the teachings herein.Examples of suitable polymers include, but are not limited to,poly(alkylene glycols), copolymers of ethylene glycol and propyleneglycol, poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(acrylic acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine, andcopolymers, terpolymers, and mixtures of any one or more of the above.

Preferably, the polymer is a polyethylene glycol or PEG, and can be inany of a number of geometries or forms, including linear chains,branched, forked, or preferably, multi-armed. PEG typically comprises—(CH₂CH₂O)_(n)—, where n ranges from about 5 to about 400, preferablyfrom about 10 to about 350, or from about 20 to about 300. In themulti-arm embodiments described here, each polymer arm typically has amolecular weight corresponding to one of the following: 200, 250, 300,400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000,7000, 7500, 8000, 9000, 10000, 12,000, 15000, 17,500, 18,000, 19,000,20,000 daltons or greater. Overall molecular weights for the multi-armedpolymer configurations described herein (that is to say, the molecularweight of the multi-armed polymer as a whole) generally correspond toone of the following: 800, 1000, 1200, 1600, 2000, 2400, 2800, 3200,3600, 4000, 6000, 8000, 12,000, 16,000, 20,000, 24,000, 28,000, 30,000,32,000, 36,000, 40,000, 48,000, 60,000 or greater. Typically, theoverall molecular weight for a multi-armed polymer of the inventionranges from about 800 to about 60,000 daltons, and preferably, fromabout 10,000 to about 60,000, or more preferably from about 20,000 toabout 40,000 daltons.

Active Agent

Typically, the active agent moiety is a small molecule anticancer agentpossessing a molecular weight of less than about 1000. In yet additionalembodiments, the small molecule drug possesses a molecular weight ofless than about 800, or even less than about 750. In yet anotherembodiment, the small molecule drug possesses a molecular weight of lessthan about 500 or, in some instances, even less than about 300.Preferred active agent moieties include anticancer agents. Particularlypreferred are oncolytics such as those having at least one hydroxylgroup.

One preferred class of active agents are the camptothecins. In oneembodiment, a camptothecin for use in a prodrug or method providedherein corresponds to the structure:

wherein R₁-R₅ are each independently selected from the group consistingof hydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl;alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;substituted amino (e.g., monoalkylamino and dialkylamino);hydroxcarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino;carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; —C(R₇)═N—(O)_(i)—R₈wherein R₇ is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, andR₈ is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C(O)O—wherein R₉ is halogen, amino, substituted amino, heterocycle,substituted heterocycle, or R₁₀—O—(CH₂)_(m)— where m is an integer of1-10 and R₁₀ is alkyl, phenyl, substituted phenyl, cycloalkyl,substituted cycloalkyl, heterocycle, or substituted heterocycle; or R₂together with R₃ or R₃ together with R₄ form substituted orunsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy; R₆ is H orOR′, wherein R′ is alkyl, alkenyl, cycloalkyl, haloalkyl, orhydroxyalkyl; and L is one exemplary site of attachment to a modifyingmoiety to provide the prodrug structure.

In one particular embodiment, D is irinotecan, where the H on the20-position hydroxyl is absent in the final multi-armed prodrugconjugate.

More particularly, the anticancer agent may fall into one of a number ofstructural classes, including but not limited to small molecules,oligopeptides, protein mimetics, fragments, or analogues, steroids,nucleotides, oligonucleotides, and the like. Preferably, an active agentfor use herein possesses a free hydroxyl, carboxyl, thio, amino group,or the like (i.e., “handle”) suitable for modification to provide thedesired prodrug. Preferably, the anticancer agent possesses at least onefunctional group suitable for forming a hydrolyzable linkage for forminga prodrug.

Alternatively, the drug is modified by introduction of a suitable“handle”, preferably by conversion of one of its existing functionalgroups to a functional group suitable for formation of a hydrolyzablecovalent linkage. Ideally, such a modification should not adverselyimpact the therapeutic effect or activity of the active agent to asignificant degree. That is to say, any modification of an active agentto facilitate its conversion to a prodrug should result in no greaterthan about a 30% reduction of its bioactivity relative to the knownparent active agent prior to modification.

Preferred anticancer agents include topoisomerase inhibitors such as butnot limited to irinotecan, topotecan, camptothecin, and lamellarin D,and microtubule inhibitors such as but not limited to vincristine,vinblastine, vinflunine, and docetaxel. Additional anticancer agentsinclude altretamine, bleomycin, capecitabine, carboplatin, carmustine,cladribine, cisplatin, cyclophosphamide, cytarabine, dacarbazine,dactinomycin, doxorubicin, imatinib, etoposide, fludarabine,fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide,methotrexate, mitomycin, mitotane, mitoxantrone, and paclitaxel.

The above exemplary drugs are meant to encompass, where applicable,analogues, agonists, antagonists, inhibitors, isomers, polymorphs, andpharmaceutically acceptable salt forms thereof.

As described previously, one preferred class of active agents is thecamptothecins. The term “camptothecin compound” as used herein includesthe plant alkaloid 20(S)-camptothecin, as well as pharmaceuticallyactive derivatives, analogues and metabolites thereof. Examples ofcamptothecin derivatives include, but are not limited to,9-nitro-20(S)-camptothecin, 9-amino-20(S)-camptothecin,9-methyl-camptothecin, 9-chloro-camptothecin, 9-flouro-camptothecin,7-ethyl camptothecin, 10-methyl-camptothecin, 10-chloro-camptothecin,10-bromo-camptothecin, 10-fluoro-camptothecin, 9-methoxy-camptothecin,11-fluoro-camptothecin, 7-ethyl-10-hydroxy camptothecin (SN38),10,11-methylenedioxy camptothecin, and 10,11-ethylenedioxy camptothecin,and 7-(4-methylpiperazinomethylene)-10,11-methylenedioxy camptothecin,7-ethyl-10-(4-(1-piperdino)-1-piperdino)-carbonyloxy-camptothecin,9-hydroxy-camptothecin, and 11-hydroxy-camptothecin. Particularlypreferred camptothecin compounds include camptothecin, irinotecan, andtopotecan.

Preferred camptothecin compounds are illustrated in the structure below

wherein R₁-R₅ are each independently selected from the group consistingof hydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl;alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;substituted amino (e.g., monoalkylamino and dialkylamino);hydroxcarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino;carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; —C(R₇)═N—(O)_(i)—R₈wherein R₇ is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, andR₈ is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C(O)O—wherein R₉ is halogen, amino, substituted amino, heterocycle,substituted heterocycle, or R₁₀—O—(CH₂)_(m)— where m is an integer of1-10 and R₁₀ is alkyl, phenyl, substituted phenyl, cycloalkyl,substituted cycloalkyl, heterocycle, or substituted heterocycle; or R₂together with R₃ or R₃ together with R₄ form substituted orunsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy; and R₆ is Hor OR′, wherein R′ is alkyl, alkenyl, cycloalkyl, haloalkyl, orhydroxyalkyl.

Exemplary substituting groups include hydroxyl, amino, substitutedamino, halo, alkoxy, alkyl, cyano, nitro, hydroxycarbonyl,alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonylamino, aryl (e.g.,phenyl), heterocycle, and glycosyl groups.

Other preferred active agents include platins, oxymorphone analogues,steroids, quinolones, isoquinolones, and fluoroquinolones, andnucleosides and nucleotides.

C. Pharmaceutical Compositions

The invention provides pharmaceutical formulations or compositions, bothfor veterinary and for human medical use, which comprise one or moreprodrugs of the invention or a pharmaceutically acceptable salt thereof,typically with one or more pharmaceutically acceptable carriers, andoptionally any other therapeutic ingredients, stabilizers, or the like.The carrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and not undulydeleterious to the recipient thereof. The compositions may also includepolymeric excipients/additives or carriers, e.g., polyvinylpyrrolidones,derivatized celluloses such as hydroxymethylcellulose,hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (apolymeric sugar), hydroxyethylstarch (HES), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin andsulfobutylether-β-cyclodextrin), polyethylene glycols, and pectin. Thecompositions may further include diluents, buffers, binders,disintegrants, thickeners, lubricants, preservatives (includingantioxidants), flavoring agents, taste-masking agents, inorganic salts(e.g., sodium chloride), antimicrobial agents (e.g., benzalkoniumchloride), sweeteners, antistatic agents, surfactants (e.g.,polysorbates such as “TWEEN 20” and “TWEEN 80”, and pluronics such asF68 and F88, available from BASF), sorbitan esters, lipids (e.g.,phospholipids such as lecithin and other phosphatidylcholines,phosphatidylethanolamines, fatty acids and fatty esters, steroids (e.g.,cholesterol)), and chelating agents (e.g., EDTA, zinc and other suchsuitable cations). Other pharmaceutical excipients and/or additivessuitable for use in the compositions according to the invention arelisted in 19^(th) “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), and in the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andin “Handbook of Pharmaceutical Excipients”, Third Ed., Ed. A. H. Kibbe,Pharmaceutical Press, 2000.

The prodrugs of the invention may be formulated in compositionsincluding those suitable for oral, rectal, topical, nasal, ophthalmic,or parenteral (including intraperitoneal, intravenous, subcutaneous, orintramuscular injection) administration. One preferred formulation isone suitable for intravenous administration. The compositions mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. All methods includethe step of bringing the active agent or compound (i.e., the prodrug)into association with a carrier that constitutes one or more accessoryingredients. In general, the compositions are prepared by bringing theactive compound into association with a liquid carrier to form asolution or a suspension, or alternatively, bring the active compoundinto association with formulation components suitable for forming asolid, optionally a particulate product, and then, if warranted, shapingthe product into a desired delivery form. Solid formulations of theinvention, when particulate, will typically comprise particles withsizes ranging from about 1 nanometer to about 500 microns. In general,for solid formulations intended for intravenous administration,particles will typically range from about 1 nm to about 10 microns indiameter. Particularly preferred are sterile, lyophilized compositionsthat are reconstituted in an aqueous vehicle prior to injection.

A preferred formulation is a solid formulation comprising a multi-armpolymer prodrug where the active agent is irinotecan. The solidformulation comprises sorbitol and lactic acid, and is typically dilutedwith 5% dextrose injection or 0.9% sodium chloride injection prior tointravenous infusion.

The amount of prodrug in the formulation will vary depending upon thespecific anticancer agent employed, its activity, the molecular weightof the prodrug, and other factors such as dosage form, target patientpopulation, and other considerations, and will generally be readilydetermined by one skilled in the art. The amount of prodrug in theformulation will be that amount necessary to deliver a therapeuticallyeffective amount of the anticancer compound to a patient in need thereofto achieve at least one of the therapeutic effects associated with thecompound, e.g., treatment of cancer. In practice, this will vary widelydepending upon the particular conjugate, its activity, the severity ofthe condition to be treated, the patient population, the stability ofthe formulation, and the like. Compositions will generally containanywhere from about 1% by weight to about 99% by weight prodrug,typically from about 2% to about 95% by weight prodrug, and moretypically from about 5% to 85% by weight prodrug, and will also dependupon the relative amounts of excipients/additives contained in thecomposition. More specifically, the composition will typically containat least about one of the following percentages of prodrug: 2%, 5%, 10%,20%, 30%, 40%, 50%, 60%, or more by weight.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,lozenges, and the like, each containing a predetermined amount of theactive agent as a powder or granules; or a suspension in an aqueousliquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, adraught, and the like.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the prodrug, which can beformulated to be isotonic with the blood of the recipient.

Methods

The prodrugs can be used to treat or prevent any condition responsive totreatment with the unmodified active agent in any animal, particularlyin mammals such as humans. The prodrugs are particularly useful aschemotherapeutics, e.g., in the treatment of cancer.

The prodrugs of the invention are particularly useful as anticanceragents, i.e., have been shown to be effective in releasing an activemoiety in vivo, where the effective half life in humans of the activemoiety (which may be an active metabolite) is greater than 50 hours.Preferably, the effective half life is greater than 7 days, or 10 days,or 14 days, or 20 days, or 21 days, or 25 days, or 28 days, or 30 days,or 35 days, or 40 days, or 45 days, or 49 days, or 50 days, or 60 days,or 70 days, or 80 days, or 90 days, or 100 days.

The prodrugs of the invention, in particular, those where the smallmolecule drug is an anticancer agent such as a camptothecin compound asdescribed herein or other oncolytic, are useful in treating solid typetumors such as breast cancer, ovarian cancer, colon cancer, gastriccancer, malignant melanoma, small cell lung cancer, non-small cell lungcancer, thyroid cancers, kidney cancer, cancer of the bile duct, braincancer, cervical cancer, maxillary sinus cancer, bladder cancer,esophageal cancer, Hodgkin's disease, adrenocortical cancer, and thelike. Additional cancers treatable with the prodrugs and methodsprovided herein include lymphomas, leukemias, rhabdomyosarcoma,neuroblastoma, and the like. As stated above, the prodrugs areparticularly effective in targeting and accumulating in solid tumors.The prodrugs are also useful in the treatment of HIV and other viruses.

The prodrugs have also been shown to be particularly advantageous whenused to treat patients having cancers shown to be refractory totreatment with one or more anticancer agents (see, e.g., Tables III andIV in Example 2). As shown in Example 2, the exemplary prodrugs providedherein exhibit anti-tumor activity in subjects whose cancer waspreviously shown to be resistant to treatment with 1, or 2, or 3, or 4,or 5 or more anticancer agents. That is to say, in prior treatments,progression of the cancer was observed, while upon administration of atherapeutically effective amount of the exemplary prodrug, compound I,anti-tumor activity was observed, either by virtue of partial tumorregression, arrestment of tumor growth, or by evidence provided by oneor more biomarkers.

Methods of treatment comprise administering to a mammal in need thereof,e.g., a human, a therapeutically effective amount of a composition orformulation containing a prodrug as described herein.

Additional methods include treatment of (i) metastatic breast cancerthat is resistant to anthracycline and/or taxane based therapies, (ii)platinum-resistant ovarian cancer, (iii) metastatic cervical cancer, and(iv) colorectal cancer in patients with K-Ras mutated gene status.

In treating metastatic breast cancer, a prodrug as provided herein isadministered to a patient with locally advanced metastatic breast cancerat a therapeutically effective amount, where the patient has had no morethan two prior (unsuccessful) treatments with anthracycline and/ortaxane based chemotherapeutics.

For treating platinum resistant ovarian cancer, a prodrug as providedherein is administered to a patient with locally advanced or metastaticovarian cancer at a therapeutically effective amount, where the patienthas shown tumor progression during platinum-based therapy. In oneparticular embodiment, the patient has had a progression-free intervalof less than six months. See, e.g., Example 3, describing the superioranti-tumor activity of the exemplary compound, 4-arm PEG-GLY-Irino-20K,in an animal model of metastatic platinum resistant ovarian cancer aswell as in a clinical phase 1 study. Efficacy of the exemplary prodrugwas clearly superior to the parent compound in both the animal model andthe preliminary phase 1 results. Moreover, the efficacious nature of4-arm PEG-GLY-Irino-20K in treating patients having undergone prioranti-cancer therapy indicates the surprising advantages of the prodrugsdescribed herein over conventional anti-tumor agents such as cis-platin.

In yet another approach, a prodrug as provided herein (e.g., such asthat in Example 1) is administered to a subject with locally advancedcolorectal cancer, where the colorectal tumor(s) has a K-Ras oncogenemutation (K-Ras mutant types) such that the tumor does not respond toEGFR-inhibitors, such as cetuximab. Subjects are those having failed oneprior 5-FU containing therapy, and are also irinotecan naïve.

In yet another aspect, the prodrug effectively achieves a metronomicdosing profile in vivo upon a single administration. See for example,FIG. 1, which illustrates the patients' exposure to the activemetabolite of irinotecan, SN-38, upon administration of the exemplaryprodrug, Compound I. As can be seen, while approved irinotecan dosingregimens of once weekly or once every 21 days result in low ornon-existent (i.e., non-detectable) plasma concentrations of SN-38 forprolonged periods of time prior to administration of a subsequent dose,administration of the instant prodrugs is effective to achieve asustained exposure to SN-38 that is similar to the exposure profileexpected for a continuous IV infusion of irinotecan, yet is achieved bydosing only once every 3 weeks.

Exemplary pharmacokinetics and pharmacodynamics of metronomic dosing ofirinotecan in metastatic colorectal cancer patients is described, e.g.,in Allegrini, G., et al., British Journal of Cancer (2008), 98,1312-1319. Exemplary metronomic doses of irinotecan explored were 1.4mg/m² per day, 2.8 mg/m² per day, and 4.6 mg/m² per day, administered bycontinuous iv infusion. The resulting plasma levels observed foririnotecan, SN-38, and SN-38 glucuronide at the various dose levelsremained above the level of detection. For example, SN-38 plasma levelsremained at around 1 ng/ml at the lowest dose for up to 63 days, whileat higher doses, plasma levels of SN-38 ranged between about 2-4 ng/mLover a dosing period of 63 days. Similarly, for irinotecan, plasmalevels over the dosing schedule ranged from about 100 ng/ml to about 250ng/ml for the lowest dose amount, and from about 10 ng/ml to about 500ng/ml over the 63 day time course observed for the higher doses. In eachcase, the plasma levels observed for the metronomic dosing scheduleswere fairly continuous and steady over time, and did not drop belowtherapeutic levels over the timecourse, in contrast to conventionaldosing in which immediately after dosing, plasma levels of theanticancer drug or active metabolite thereof begin to steadily declineover time, eventually dropping below levels of detection.

In some instances, the prodrug, when administered in vivo, provides adesired concentration-time profile to achieve a desired pharmacodynamiceffect, wherein the desired pharmacodynamic effect is different from theknown (or understood or accepted) pharmacodynamic effect of the activemoiety. For example, the prodrug is a prodrug of irinotecan, where thedesired pharmacodynamic effect is antiangiogenesis, and the knownpharmacodynamic effect is topoisomerase inhibition. Alternatively, theprodrug is a prodrug form of docetaxel, where the desiredpharmacodynamic effect is antiangiogenesis, and the knownpharmacodynamic effect is microtubule inhibition.

A therapeutically effective dosage amount of any specific prodrug willvary from conjugate to conjugate, patient to patient, and will dependupon factors such as the condition of the patient, the activity of theparticular active agent employed, the type of cancer, and the route ofdelivery.

For camptothecin-type active agents, dosages from about 0.5 to about 100mg camptothecin/kg body weight, preferably from about 10.0 to about 60mg/kg, are preferred. When administered conjointly with otherpharmaceutically active agents, even less of the prodrug may betherapeutically effective. For administration of a prodrug ofirinotecan, the dosage amount of irinotecan will typically range fromabout 50 mg/m² to about 350 mg/m².

Methods of treatment are meant to encompass both monotherapy with asingle prodrug, as well as administration of a therapeutically effectiveamount of a prodrug as described herein in conjunction with a secondanticancer agent. for example, a camptothecin type prodrugs isadministered in combination with 5-fluorouracil and folinic acid, asdescribed in U.S. Pat. No. 6,403,569.

The prodrugs of the invention may be administered once or several timesa day, preferably once a day or less, or once a week or less.Illustrative dosing regimens include dosing of a therapeutic amount ofprodrug once every 7 days, or once every 14 days, or once every 21 days,or once every 28 days. The duration of the treatment may continue for aperiod of one month to several years. For example, the course oftreatment may extend over one month, two months, three months, fourmonths, five months, or six months, or even longer. Each dose can beadministered either by a single dose in the form of an individual dosageunit or several smaller dosage units or by multiple administration ofsubdivided dosages at certain intervals.

EXAMPLES

It is to be understood that while the invention has been described inconjunction with certain preferred specific embodiments thereof, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

Example 1 Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2Oxo-Vinylamino Acetate Linked-Irinotecan)-20K (“4-Arm PEG-GLY-Irino-20K,or Compound I)

The synthesis of 4-arm PEG-GLY-Irino-20K (Compound I) has beenpreviously described. See, e.g., Applicant's United States PatentPublication No. 2005-0112088, Example 1.

Example 2 Phase I Clinical Study in Patients having Advanced Stages ofCancer

In the Phase 1 dose-escalation trial, the safety, pharmacokinetics andanti-tumor activity of Compound I monotherapy were evaluated in 44patients with advanced solid tumors who had failed prior treatments orhad no standard treatment available to them. Patients received 90-minuteinfusions of Compound I as follows: weekly for three weeks with thefourth week off (n=32); q 14 days or every two weeks (n=6); and q 21days or every three weeks (n=6). Tumor responses were evaluatedaccording to RECIST (Response Evaluation Criteria in Solid Tumors)criteria.

Doses ranged from 58 mg/m² to 230 mg/m² in the weekly dose schedule(weekly ×3 Q4 weeks). In the every two week dose schedule (Q14 days) andevery three week dose schedule (Q21 days), doses ranged from 145 mg/m²to 170 mg/m². Tumor regression, anti-tumor activity or prolonged diseasestabilization was observed in a broad spectrum of cancer types,including breast, ovarian, cervical, bladder, non-small cell lungcancer, small cell lung cancer, adrenocortical, esophageal, maxillarysinus and Hodgkins lymphoma. Side effects of clinical significanceobserved in the first and second dose schedules were diarrhea andneutropenia, with diarrhea being the dose-limiting toxicity associatedwith Compound I. There was no significant diarrhea and neutropeniaobserved in the every three week (Q21 days) dose schedule.

TABLE 1 RESULTS OF COMPOUND I WEEKLY X3 Q4 WEEKS DRUG-RELATED TOXICITIESAS OBSERVED IN ANY COURSE Dose Median # Level Courses Diarrhea DiarrheaNeutropenia Neutropenia mg/m² Enrolled (range) G3 G4 G3 G4 58 3 2 (2-6)0 0 0 0 115 6 5+ (1-6) 1 0 1 0 145 6 1 (0.33-4) 2 0 3 0 173 14 2.5(0.67-6) 7 0 3 0 230 3 0.87 (0.33-1.87) 3 0 1 0 Total 32 13 0 8 0 Doseescalation continued per protocol until the MTD (maximum tolerated dose)of 230 mg/m² was reached. All 3 patients at this dose had dose limitingG3 diarrhea. The lower dose cohort of 173 mg/m² was therefore expanded,delayed observation of G3 diarrhea (7/14 patients) necessitatedexpansion of the next lower cohort. In the next lower dose cohort at 145mg/m², 2/6 patients had coexistent G3 diarrhea and G3 neutropenia incourse 1. Therefore, the next lower dose cohort was further evaluated.In the next lower dose cohort of 115 mg/m², one patient's G3 diarrheacoincident to anti-diarrheal medication noncompliance was not considereddose limiting. Except for this one patient, the 115 mg/m² dose was welltolerated and subsequently declared as the RP2D for the weekly x3 q4weeks schedule.

On the weekly ×3q4 weeks, the MTD/RP2D was 115 mg/m² and toxicity wasmanageable. Visual disturbances (floaters) noted in 13 of 32 patientswere transient and self-limiting.

TABLE 2 RESULTS OF COMPOUND I WEEKLY Q14 DAYS DRUG-RELATED TOXICITIES ASOBSERVED IN ANY COURSE Dose Median # Level Courses Diarrhea DiarrheaNeutropenia Neutropenia mg/m³ Enrolled (range) G3 G4 G3 G4 145 3 4 (3-7)3 0 0 1 170 3 4 (3-7) 1 0 1 0 195 3 3 (2-4) 0 0 0 0 220 1 3 0 0 0 0 2patients had G2 alopecia. 1 patient had transient, self-limited visualdisturbances (floaters) associated with dosing.

TABLE 3 RESULTS OF COMPOUND I WEEKLY Q21 DAYS - ONGOING DRUG-RELATEDTOXICITIES AS OBSERVED IN ANY CYCLE Dose Median # Level Courses DiarrheaDiarrhea Neutropenia Neutropenia mg/m³ Enrolled (range) G3 G4 G3 G4 1453   2 (1-4) 0 0 0 0 170 3 3.7 (2-6) 0 0 0 0 195 3 ongoing 0 0 0 0 220 3ongoing 0 0 0 1 245 3 ongoing 1 0 0 0 1 patient had transient,self-limited visual disturbances (floaters) associated with dosing.

Sustained SN-38 Exposure and Half-life:

Serial plasma concentrations of Compound I, irinotecan, activemetabolite SN38 and SN38-glucuronide were quantified by LC-MS/MS atmultiple time points. SN38 AUCs after Compound I administration wereestimated for each patient with concentration-time profiles as predictedwith a 2-compartment exponential-error population PK model implementedusing Monolix v2.3. SN38 AUCs after patient specific dose/schedule/# ofdoses of irinotecan administered were predicted using a population PKmodel for irinotecan administration (Xie, R., et al., JCO. 2002:20(15),3293-33-1).

Compound I exhibited extended pharmacokinetics in the Phase 1 trial.Specifically, administration of Compound I produced an increase incumulative SN38 exposure that was up to several-fold (4.4-fold) higherthan the exposure previously reported with irinotecan. SN38, atopoisomerase I inhibitor, is the active metabolite of irinotecan. Thelong 50-day half life of SN-38 following administration of Compound Iresults in plasma SN38 concentrations that are significantly moresustained between doses than are possible with irinotecan.

TABLE 4 SN-38 CL_(TOT) BY DRUG AND DOSING REGIMEN CL = Dose, CumulativeAUC Dose/ Drug mg/m² Schedule Dose (0-4000) AUC Irino 350 q 3 Wks 28004508 0.621 Irino 125 q Wk × 4 2000 3220 0.621 Compound I 50 q Wk × 3 7505227 0.143 Compound I 200 q 3 Wks 1600 10842 0.148

Plasma SN38 Cmax and AUC values increased in a linear fashion.Inter-patient variability in SN38 CL and V1 parameters ranged from ˜40to ˜60%, similar to that reported for SN38 after irinotecanadministration (Xie, R., et al., JCO. 2002:20(15); 3293-3301).

SN-38 CL_(tot) is four times less in comparison to irinotecan followingadministration of Compound I. Even when dosed once every three weeks,the SN-38 CP-T profile appears similar to one achieved by continuousinfusion.

See FIG. 1 which demonstrates sustained exposure to the activemetabolite, SN-38, upon administration of Compound I, versusadministration of irinotecan (Camptosar®). Camptosar® is cleared rapidlyrelative to Compound I—with plasma concentrations dropping below thelevel of detection by approximately 7 days (1 week) post-dosing. Incontrast, administration of Compound I achieves prolonged exposure totherapeutically effective levels of SN-38 throughout each course ofdosing, with plasma concentrations of SN-38 never observed to fall belowthe level of detection—even when measured at 21 days (3 weeks)post-dose. Sustained plasma SN38 concentrations are achieved uponadministration of Compound I, even in the fourth (non-dosed) weeks of aweekly ×3q4 weeks schedule. Roughly, it appears that upon dosing withCompound I, plasma concentrations of SN-38 remained above about 0.5ng/mL, even at 21 days post-dosing! Cumulative SN38 exposures wereapproximately 2-fold greater than those predicted for irinotecan dosedat equivalent doses and schedules (mean 1.6, SD 0.8, min 0.6, max 4.4).Upon administration of Compound I, the effective half life of SN-38 wascalculated to be approximately 50 days, while in contrast, the half-lifeof SN-38 achieved via administration of Camptosar® has been reported tobe 47 hours.

FIG. 1 strikingly illustrates that while the active metabolite ofCamptosar® is cleared relatively rapidly after administration(essentially dropping to below detection limits within 7 days followingdosing), the plasma concentrations of the active metabolite of CompoundI remain at detectable and even therapeutic levels over the course ofduration of treatment—to at least 21 days post-dosing. As can be seen,the active metabolite of Compound I was determined to have a half-lifeof approximately 50 days, while the half life of the active metaboliteof Camptosar® was determined to be approximately 47 hours.

TABLE 5 HALF-LIVES Population Estimate in Responding In RespondingParameter Analyte Patients Patients T_(1/2) λ_(z) (days) Compound I  7.4days  6.8-8.3 days Irinotecan 24.8 days 19.2-34.1 days SN-38 49.1 days14.4-98.3 days

The differences in half-life and sustained plasma concentrations werestriking between the two compounds, and demonstrated the surprising andunexpected clear superiority of Compound I as an anticancer agent.

Anti-Tumor Activity

Although the study was designed to evaluate safety, a notable number ofpatients surprisingly responded favorably to treatment by exhibitingeither a cessation of tumor growth or a reversal (regression) of tumorsize, where treatment with prior anti-cancer agents had failed. SeeTable 6 below.

TABLE 6 SIGNIFICANT ANTI-TUMOR ACTIVITY INCLUDES PARTIAL RESPONSES ANDADDITIONAL HIGH BIOMARKER ACTIVITY IN PHASE 1 STUDY Dose, mg/m²Progression Dosing Anti-tumor # Courses to Observed with Prior TumorSchedule Activity Response Agents Ovarian 145 PR: RECIST 2Investigational agent, q 14 days ↓ 48% docetaxel, carboplatin, ↓ CA-125tamoxifen, paclitaxel, 1900 U/ml cisplatin →150 U/ml Breast (triple 170PR: RECIST 2 Bevacizumab, Doxil ®, negative) q 21 days ↓ 41% docetaxel,cisplatin, gemcitabine, doxorubicin, cyclophosphamide Cervical 173 PR:RECIST 2 Cisplatin, gemcitabine, x3 q 4 ↓ 53% pegaspargase weeks (on aclinical trial) Maxillary sinus 170 PR: RECIST 2 None (neuroendocrine) q14 days ↓ 35% 4 and RECIST ↓ 45% Bladder 145 PR: RECIST 2 Cisplatin,gemcitabine, (transitional cell q 14 days ↓ 35% 4 paclitaxel with smallcell and RECIST features) ↓ 53% Small cell lung  58 PR: RECIST 2Investigational agent, x3 q 4 ↓ 56% gemcitabine, weeks topotecan,carboplatin, etoposide, cisplatin Non small cell 145 PR: RECIST 1Pemetrexed, docetaxel, lung x3 q 4 ↓ 52% carboplatin weeks PR = partialregression

Significant anti-tumor activity was observed in all dose schedules ofthe study with partial responses observed in seven out of 44 totalpatients (16 percent) in the trial. Of the 44 patients in total from alldose schedules, 13 patients exhibited anti-tumor activity. Sevenpatients, or 16 percent, had partial responses (greater than 30 percenttumor regression per RECIST), and six patients, or 12 percent, had otherevidence of anti-tumor activity (tumor regression by more than 15percent but less than 30 percent per RECIST, or significant biomarkerevidence). Repeat evidence of anti-tumor activity was observed in anumber of tumor settings, including breast and ovarian.

TABLE 7 BIOMARKER ACTIVITY IN PHASE 1 STUDY Dose, mg/m² # CoursesProgression Dosing Anti-tumor to Observed with Prior Tumor ScheduleActivity Response Agents Breast (triple 145 CT scan ↓ 20% 2 doxorubicin,negative) q 14 days cyclophosphamide, XRT, Investigational agent,capecitabine plus bevacizumab Breast (triple 170 ↓ CA 27.29 2 docetaxel,cisplatin, negative) q 14 days 837 U/ml → gemcitabine, 383 U/mldoxorubicin, cyclophosphamide, XRT, oxaliplatin Ovarian 173 ↓ CA-125 0.6cisplatin, gemcitabine, x3 q 4 2558 U/ml → carboplatin, paclitaxel weeks518 U/ml Esophageal  58 ↓ CEA 2 cisplatin, carboplatin, x3 q 4 36 ng/ml→ 14 ng/ml paclitaxel, 5-FU weeks PET scan response Hodgkin's 115 CTscant↓ 14% 2 ifosfamide, carboplatin, Disease x3 q 4 ↓ LDH etoposide,topotecan, weeks paclitaxel, doxorubicin, dacarbazine, bleomycin,vinblastine, procarbazine, prednisone

Example 3 Nonclinical and Phase 1 Clinical Anti-Tumor Activity inPlatinum-Resistant Ovarian Cancers

This study was undertaken to investigate the nonclinical and clinicalanti-tumor activity of 4-arm PEG-GLY-Irino-20K (Compound I) inmetastatic platinum resistant ovarian cancer.

Non-Clinical Protocol:

Mice bearing A2780 ovarian tumors that are minimally responsive tocisplatin received Compound I or irinotecan in 3 weekly doses of 50,100, or 150 mg/kg. Anti-tumor efficacy was evaluated based on tumorgrowth delay (TGD) in mice and response rate in mice and humans.

Specifically, A2780 tumor cells were grown in vitro and implanted s.c.as a cell suspension in 9 to 10 weeks old female nude mice (nu/nu,Harlan) weighing 18-26 g. When the tumor volume reached an average of146 mm³, mice (n=10/treatment) were randomized into the treatment groupsand treated with 3 weekly intravenous injections of irinotecan orCompound I, or 3 weekly intra-peritoneal administrations of cisplatin orcarboplatin.

Animals were weighed and monitored twice weekly, and tumor volumes weremeasured until an endpoint was met (2000 mm³ or 60 days). Anti-tumorefficacy was evaluated based on tumor growth delay and response rate orpartial or complete regression (PR or CR). In a PR, the tumor volume was50% or less of its Day 1 volume for three consecutive measurements, andequal to or greater than 14 mm³ for one or more of these threemeasurements. In a CR, the tumor volume was less than 14 mm³ for threeconsecutive measurements. An animal with a CR response at terminationwas additionally classified as tumor-free survivior (TFS). A Student'sT-test was performed to calculate statistical significance (p<0.05).

Clinical Protocol:

In the phase 1 study of Compound I, 5 patients with ovarian cancer wereenrolled in weekly ×3 q4w, q14d and q21d regimens. Patients withmeasurable disease were assessed for tumor response using RECIST 1.0every other cycle.

Specifically, in an open-label, dose-escalation, multicenter Phase 1study, patients with advanced solid tumors whose tumors had failed priortreatments were enrolled. The study enrolled a total of 72 patients,five of whom had ovarian cancer. Patients received 90-minute infusionsof NKTR-102 on either a weekly ×3 every 4 weeks (qw×3 q4w), every 14days (q14d), or every 21 days (q21d) schedule. Patients were evaluatedfor safety, pharmacokinetics and evidence of anti-tumor activity (byRESIST 1.0 guidelines).

Results

Mice: The response summary is provided in the table provided as FIG. 3.Control tumors grew rapidly and uniformly to the 2000 mm³ endpoint in amedian of 14 days. Irinotecan administered at 50, 100, and 150 mg/kgresulted in TGD of 12, 15, and 16 days, respectively, with one partialresponse (PR) at the highest dose. Compound I when administered atirinotecan-equivalent doses resulted in TGDs of 33, 32, and 34 days,respectively, with 100% regression response rate (PRs+CRs) in eachgroup. Increasing Compound I doses were associated with increasednumbers of CR responses (5, 8, and 9 CRs, respectively). Compound I wassuperior to the equivalent irinotecan dose at all doses tested and thelowest dose of Compound I was superior to the highest irinotecan dose.

In summary, Compound I shows superior activity compared to irinotecan inthe A2780 ovarian tumor model, inducing a 100% response rate at alldoses and dose-related increases in CRs versus PRs. See FIG. 2. Controltumors grew to the 2000 mm3 endpoint in a median of 14 days,establishing a maximum possible tumor growth delay of 46 days (329%) forthe 60 day study. Cisplatin and carboplatin were inactive. Further,Compound I was well tolerated, while irinotecan resulted in acuteeffects immediately after dosing (tremors, hypoactivity, ataxia,pilo-erection, increased stool production).

Humans:

In the phase 1 clinical study, tumor response could be assessed in 2 of5 patients. Of these two patients, one patient receiving 145 mg/m² q14(sixth line) had an unconfirmed partial response (37% reduction intarget lesions) but terminated from the study prior to confirmation, andone patient on the weekly regimen receiving 172.5 mg/m² had a mixedresponse that included a 21% reduction in target lesions.

The foregoing results indicate the superior nature of exemplary prodrug,4-arm PEG-GLY-Irino-20K (Compound I), in comparison not only toirinotecan, but also to other forms of cancer therapy, namely cis-platinamd carboplatin. The non-clinical data is striking in its results, anddemonstrates the surprising efficacy and tolerability of the compoundsand methods provided herein.

Many modifications and other embodiments will come to mind to oneskilled in the art to which this disclosure pertains having the benefitof the teachings presented in the foregoing description. Therefore, itis to be understood that the disclosure is not to be limited to thespecific embodiments disclosed and that modifications and otherembodiments are intended to be included within the scope of theteachings herein. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

It is claimed:
 1. A method of achieving sustained exposure to SN-38 viaadministration of irinotecan to a mammalian subject, the methodcomprising administering via a non-continuous dosing regimen to amammalian subject having breast cancer, a therapeutically effectiveamount of a pharmaceutical composition comprising a prodrugcorresponding to structure (I):

or a pharmaceutically acceptable salt thereof, wherein thenon-continuous dosing regimen comprises administering the pharmaceuticalcomposition no more frequently than once every seven days for at leasttwo dosings, to thereby maintain sustained therapeutic levels of greaterthan 0.5 ng/mL of SN-38 in plasma in the subject for at least 21 daysbetween dosings.
 2. The method of claim 1, wherein the dosing regimencomprises administering the pharmaceutical composition once every 21days.
 3. A method for achieving extended therapeutic efficacy ofirinotecan, the method comprising administering to a mammalian subjecthaving breast cancer, a therapeutically effective amount of apharmaceutical composition comprising a prodrug corresponding tostructure (I):

or a pharmaceutically acceptable salt thereof, wherein the administeringcomprises administering the composition at a frequency of between onceevery 7 days to once every 30 days for at least two dosings, to therebyachieve for each of the at least two dosings an elimination half-life inplasma of SN-38 that exceeds 750 hours and greater than 0.5 ng/mL ofSN-38 in plasma in the subject for at least 21 days.
 4. The method ofclaim 3, wherein the overall nominal average molecular weight of theprodrug ranges from about 10,000 to about 60,000 daltons.
 5. The methodof claim 3, wherein the administering occurs at a frequency of onceevery 7 days.
 6. The method of claim 1, wherein the administeringcomprises administering to the subject a dosage amount of irinotecanranging from about 70 mg/m² to about 300 mg/m².
 7. The method of claim1, wherein the administering is effective to prevent tumor growth asmeasured from the start of treatment.
 8. The method of claim 1, whereinthe administering is effective to result in tumor size regression. 9.The method of claim 3, wherein the administering occurs at a frequencyof once every 14 days.
 10. The method of claim 3, wherein theadministering occurs at a frequency of once every 21 days.
 11. Themethod of claim 3, wherein the administering occurs at a frequency ofonce every 28 days.
 12. The method of claim 1, wherein the administeringoccurs for 2-6 dosings.
 13. The method of claim 3, wherein theadministering occurs for 2-6 dosings.